Guide to Presaturation Experiments

Very often need to remove large, unwanted peaks from a spectrum, e.g., running samples in H₂O so that slowly exchangeable protons can be observed. Note that if your protons exchange at a sizeable rate (> 0.1 s^-1) with the solvent resonance you want to remove, presaturation is inappropriate and an experiment such as WATERGATE such be used.

Why?

• Eliminate dynamic range problems to improve signal to noise
• Cosmetic improvement
• Save cash on expensive deuterated solvents e.g. CD₂Cl₂ and THF-d₈

If you wish to save on deuterated solvent, use a protio solvent with just one signal, e.g., CH₂Cl₂, C₆H₆, 1,4-dioxane (can often be used in place of THF) as presaturating multiple signals is generally much less effective and slightly more difficult to set up. If possible a small amount of deuterated solvent (e.g. 10 % C₆D₆, D₂O or acetone-d₆ which are relatively cheap) should be added to facilitate locking and shimming. If not, spectra can be run unlocked by manually activating the sweep off and shimming on the fid.

When?

If your sample is very concentrated, you may not need to use presaturation with a protio solvent. If the solvent is less than 100 times bigger than the smallest peak of interest, there may be little need for presaturation as only a cosmetic improvement will be obtained. The real need for presaturation arises when signals cannot be amplified enough to register sufficient bits on the ADC due to the presence of the large signal preventing the receiver gain from being increased to a sufficient level.

Theory

A low power (rf field strength ~ tens of Hz) continuous wave irradiation is applied at the frequency of the signal that is to be reduced in intensity, typically for 1-3 seconds prior to acquisition, thus saturating the resonance.

In practice, on certain machines (the DMX/DPX's) there may be no physical difference between the "observe" and "decoupler" channels, i.e. the source (rf amplifier) generating the low power irradiation and the source of the high power pulse is the same. To run this experiment two parameters need to be set, the decoupler frequency, which can be O1 or O2 and the irradiation power. Too little rf power means the presaturated peak is not completely removed. Too much power can lead to large negative or dispersive "peaks" at the presaturation frequency and severe attenuation of nearby resonances. Remember that rf powers are in attenuation units on Bruker spectrometers. Adding 6 dB halves the power output.

Presaturation on the DMX/DPXs

The presaturation can be done in two ways either using the O2 or the O1 frequency. Using O1 is easiest but requires that the presaturation frequency be exactly in the center of the spectrum which is a drawback when the solvent resonance is towards one extreme of the chemical shift range but may give slightly better suppression. Using O2 requires slightly more setting up.

Presat at O1

Uses pulse program zg0pr shown below:

;zg0pr
;avance-version
;1D sequence with f1 presaturation
;using p0 for any flip angle

#include <Avance.incl>

"d12=20u"
"d13=3u"

1 ze
2 d12 pl9:f1
d1 cw:f1
d13 do:f1
d12 pl1:f1
p0 ph1
go=2 ph31
wr #0
exit

ph1=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1

;pl1 : f1 channel - power level for pulse (default)
;pl9 : f1 channel - power level for presaturation
;p0 : for any flip angle
;d1 : relaxation delay; 1-5 * T1
;d12: delay for power switching [20 usec]
;d13: short delay [3 usec]

Explicit procedure for presat at O1 on DPXs

(Commands that are to be typed are in quotation marks. Writing in curly brackets {} indicates a button on the XWINNMR display.)

1. Start with a routine set up. i.e. Shim on the lock if possible. Set normal ¹H parameters, solvent offset and spectral window as per normal. Run a 1 scan spectrum. Process and reference as normal.
2. Set O1 to match the frequency to be removed (the top of the big peak!)- this will be both the observe and decoupler frequency. Use mouse to go into{utilities}, then select define O1 by clicking the left mouse in {O1}. Place cursor on top of peak and click middle mouse button. Change 'sw' (spectral width) if necessary to include all peaks of interest
3. Type 'presato1' This runs a macro which installs some defaults.
   The defaults are:
   pulprog zg0pr - the appropriate pulse program
   d1 2s - presaturation "pulse" of two seconds duration
   pl9 70dB - presat power level
   ds 2 - two dummy scans
4. Change defaults at this stage if desired (the 'ased' command is useful for this)
5. Use 'gs' or 'rga' to set the receiver gain (rg).
6. Acquire with 'zg'
7. Decrease 'pl9' if decoupler power too low and reacquire. 'pl9' should never be less than 40 dB. Increase 'pl9' if decoupler power too high and reacquire.
   

Presat at O2

Uses pulse program zg0f2pr shown below:

;zg0f2pr
;avance-version
;1D sequence with f2 presaturation
;using p0 for any flip angle

#include <Avance.incl>

"d11=30m"
"d13=3u"

1 ze
d11 pl14:f2
2 d1 cw:f2
d13 do:f2
p0 ph1
go=2 ph31
wr #0
exit

ph1=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1

;pl1 : f1 channel - power level for pulse (default)
;pl14: f2 channel - power level for cw/hd decoupling
;p0 : for any flip angle
;d1 : relaxation delay; 1-5 * T1
;d11: delay for disk I/O [30 msec]
;d13: short delay [3 usec]

Explicit procedure for presat at O2:

1. Start with a routine set up. i.e. Shim on the lock if possible. Set parameters, solvent offset and spectral window as per normal. Run a 1 scan spectrum. Process and reference as normal.
2. Type 'edasp'. Click the button labelled 'off' under the 'F2' button and select 1H then click 'save' to activate proton decoupling.
3. Set O2 - saturation frequency. Expand signal to be saturated. Use mouse to go into {utilities}, then select define O2 by clicking the left mouse in {O2}. Place cursor on top of peak and click middle mouse button.
4. Type 'presato2'. This runs a macro which installs some defaults. The defaults are:
   pulprog zg0f2pr - the appropriate pulse program
   d1 2s - relaxation delay with presat
   pl14 70dB - presat power level
   ds 2 - two dummy scans
5. Change defaults at this stage if desired (the 'ased' command is useful for this)
6. Use 'gs' or 'rga' to set the receiver gain (rg).
7. Acquire with 'zg'
8. Adjust saturation power if necessary - Decrease 'pl14' if decoupler power too low and reacquire. 'pl14' should never be less than 40 dB. Increase 'pl14' if decoupler power too high and reacquire.

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